Measurement of the dissolved oxygen concentration is an important analytical task. Measurement of the rates of oxygen uptake or release can be used for the monitoring of various chemical and biological processes. Oxygen is one of the key metabolites in living cells, tissues, organisms, sub-cellular fragments, which is continuously being consumed and/or released. Monitoring of oxygen uptake/release by particular enzymes, living cells, tissues or whole organisms can provide information about their activity, metabolic status, viability, and/or physiological response, for example as a result of the action of a drug, environmental stress, toxicant, gene or effector.
Oxygen consumption can be quantified for example by measuring pressure change in the headspace of samples placed in closed test-vials (U.S. Pat. No. 5,232,839). Dissolved oxygen concentration can be measured by electrochemical oxygen sensors such as Clark-type electrode, by gas chromatography, using paramagnetic zirconium sensors or by fluorescence quenching.
Quantitation of oxygen by luminescence quenching has a number of advantages compared to other techniques. Quenched-luminescence sensing of oxygen is usually performed using dedicated oxygen-sensitive materials based on long-decay photoluminescent dyes. Such optical oxygen probes usually comprise an oxygen-sensitive dye in an appropriate quenching medium, such as plastics. U.S. Pat. Nos. 4,003,707 and 4,810,655 describe oxygen sensing systems which employ solid-state oxygen-sensitive materials based on fluorescent pyrene butyrate and phosphorescent palladium(II)- and platinum(II)-porphyrins, respectively. Oxygen probes based on fluorescent ruthenium dyes embedded in polymers such as silicon rubber (U.S. Pat. No. 5,030,420) and Pt- and Pd-complexes of porphyrin-ketones in polystyrene and other polymers (U.S. Pat. No. 5,718,842) have also been described. Such solid-state oxygen probes are usually prepared in the form of a coating or a membrane permeable for oxygen which is brought in contact with a test sample where oxygen concentration is to be determined.
Such solid-state luminescent oxygen probes and systems based thereon have been used in various assays and applications in particular in blood gas analysis, monitoring biological activity, presence of microorganisms, cellular respiration, action of drugs, toxicants and effectors on cells, or sterility for example (WO 98/15645 and U.S. Pat. No. 5,371,016). In these assays biological samples contained living microorganisms were assessed by measuring gradients of the dissolved oxygen in a special vessel, such as sealed, vial or well of a microtiter plate. These systems suffer from the following disadvantages: limited assay flexibility due to the permanent attachment of the sensitive material to solid support, significant waste of sensing materials, particularly in applications with high sample throughput, limited possibility for the user to change the amount of sensing material and assay format used, and relatively high assay cost.
Water-soluble luminescent oxygen probes have also been described. For example, Vanderkooi et al (“An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence”, J. Biol. Chem., 262 (12):5476-5482 [1987]) describe a method and apparatus for imaging of oxygen distribution in tissue using non-covalent complexes of phosphorescent meso-substituted porphyrins bound non-covalently to proteins, namely, Pd(II)-tetrakis-(4-carboxyphenyl)porphine and albumin. Similarly Vinogradov S. A. et al. (Non-invasive imaging of the distribution in oxygen in tissue in vivo using near-infrared phosphors, Biophys. J. 1996, v. 70, p. 1609-1617) describe water-soluble non-covalent complexes of Pd(II)-tetrabenzoporphyrins with serum albumin as oxygen probes for imaging. Relatively long lifetimes of these dyes result in their high sensitivity to oxygen and strong quenching in aqueous solutions. These probes are suitable for fluorescence lifetime-based detection of oxygen, however they have undefined chemical composition, and there is the possibility of binding of the dye to cells and other sample components, self-quenching of the dye and potential phototoxic action on cells.
Water-soluble phosphorescent structures on the basis of Pd-tetrakis-(4-carboxyphenyl)porphine and Pd-tetrakis-(4-carboxyphenyl)benzoporphine conjugated to multiple branched polyethyleneglycol (PEG) and polyglutamate chains have been suggested as oxygen probes (U.S. Pat. No. 5,837,865) and for cell-respirometric assays and drug screening applications (U.S. Pat. No. 6,395,555). However, such probes have complex structures (dendrimers), display heterogeneity of spectral and quenching properties, bear significant electrical charge and protonable groups. They are susceptible to various transitions and conformational changes in solutions and can also bind to sample components (e.g. albumin). All this affects spectral properties and response to oxygen of such probes (Dunphy I. et al.—Anal. Biochem., 2002, v. 310, p. 191-8; Rietveld I. B. et al, Tetrahedron, 2003, v. 59, p. 3821-3831). These probes; which emit at above 700 nm, are also difficult to measure on PMT-based fluorescence spectrometers and plate readers, which are rather insensitive in the very-near infrared spectral range.
Water-soluble fluorescent ruthenium dyes have been described for use as oxygen-sensitive probes (U.S. Pat. No. 6,306,661). These oxygen probes have much shorter emission lifetimes than the porphyrins (only few microseconds), and they are not very compatible with standard time-resolved fluorescence plate readers which typically have time resolution of above 10 microseconds. Due to polycyclic aromatic structure and relatively low molecular weight, they can be cell-permeable and toxic to the cells. The sensitivity to oxygen for these oxygen probes is not as good as for the porphyrin-based probes.
An improved and more efficient method for measuring dissolved oxygen concentration in chemical and biological processes would be very useful in a wide range of applications.